ADAMTS-13: A Prognostic Biomarker for Portal Vein Thrombosis in Japanese Patients with Liver Cirrhosis.

Portal vein thrombosis (PVT), one of the most prevalent hepatic vascular conditions in patients with liver cirrhosis (LC), is associated with high mortality rates. An imbalance between a disintegrin-like metalloproteinase with thrombospondin type-1 motifs 13 (ADAMTS-13) enzyme and von Willebrand factor (VWF) is responsible for hypercoagulability, including spontaneous thrombus formation in blood vessels. Herein, we aimed to identify potential prognostic and diagnostic biomarkers in Japanese patients with LC and PVT. In total, 345 patients were divided into two groups: 40 patients who developed PVT (PVT group) and 305 who did not develop PVT (NPVT group). Among the 345 patients with LC, 81% (279/345) were deemed ineligible due to the presence of preventive comorbidities, active or recent malignancies, and organ dysfunction. The remaining 66 patients were divided into two groups: the PVT group (n = 33) and the NPVT group (n = 33). Plasma ADAMTS-13 activity (ADAMTS-13:AC) and the vWF antigen (VWF:Ag) were measured using enzyme-linked immunosorbent assays. Contrast-enhanced, three-dimensional helical computed tomography (CT) was used to detect and characterize PVT. ADAMTS-13:AC was significantly lower in the PVT group than in the NPVT group. No significant differences in plasma vWF:Ag or liver stiffness were observed between the two groups. ADAMTS-13:AC of <18.8 was an independent risk factor for PVT on multivariate analyses (odds ratio: 1.67, 95% confidence interval: 1.21-3.00, p < 0.002). The receiver operating characteristic analysis of ADAMTS-13:AC revealed an area under the curve of 0.913 in PVT detection. Patients with PVT having ADAMTS-13:AC ≥18.8 (n = 17) had higher albumin levels and better prognoses than those with ADAMTS-13:AC <18.8 (n = 16). No significant correlations of ADAMTS-13:AC levels with either fibrin degradation product or D-dimer levels were observed. ADAMTS-13:AC levels could be potential diagnostic and prognostic biomarkers for PVT in Japanese patients with LC.

Combination of a synthetic retinoid and a DNA demethylating agent induced differentiation of neuroblastoma through retinoic acid signal reprogramming.

BACKGROUND: The CpG island methylator phenotype of neuroblastoma (NBL) is strongly associated with poor prognosis and can be targeted by 5-aza-2'-deoxycytidine (5-aza-dC). Differentiation therapy is a standard maintenance therapy for high-risk NBLs. However, the in vivo effect of tamibarotene, a synthetic retinoic acid, and the efficacy of its combination with 5-aza-dC have not been studied. Here, we conducted a preclinical study to assess the in vivo tamibarotene effect and the combination. METHODS: Treatment effects were analysed by in vitro cell growth and differentiation state and by in vivo xenograft suppression. Demethylated genes were analysed by DNA methylation microarrays and geneset enrichment. RESULTS: Tamibarotene monotherapy induced neural extension and upregulation of differentiation markers of NBL cells in vitro, and tumour regression without severe side effects in vivo. 5-Aza-dC monotherapy suppressed tumour growth both in vitro and in vivo, and induced demethylation of genes related to nervous system development and function. Pre-treatment with 5-aza-dC in vitro enhanced upregulation of differentiation markers and genes involved in retinoic acid signaling. Pre-treatment with 5-aza-dC in vivo significantly suppressed tumour growth and reduced the variation in tumour sizes. CONCLUSIONS: Epigenetic drug-based differentiation therapy using 5-aza-dC and TBT is a promising strategy for refractory NBLs.

Combined effect of a farnesoid X receptor agonist and dipeptidyl peptidase-4 inhibitor on hepatic fibrosis.

AIM: Non-alcoholic steatohepatitis (NASH) has a broad clinicopathological spectrum (inflammation to severe fibrosis). The farnesoid X receptor agonist obeticholic acid (OCA) ameliorates the histological features of NASH; satisfactory antifibrotic effects have not yet been reported. Here, we investigated the combined effects of OCA + a dipeptidyl peptidase-4 inhibitor (sitagliptin) on hepatic fibrogenesis in a rat model of NASH. METHODS: Fifty Fischer 344 rats were fed a choline-deficient L-amino-acid-defined (CDAA) diet for 12 weeks. The in vitro and in vivo effects of OCA + sitagliptin were assessed along with hepatic fibrogenesis, lipopolysaccharide-Toll-like receptor 4 (TLR4) regulatory cascade and intestinal barrier function. Direct inhibitory effects of OCA + sitagliptin on activated hepatic stellate cells (Ac-HSCs) were assessed in vitro. RESULTS: Treatment with OCA + sitagliptin potentially inhibited hepatic fibrogenesis along with Ac-HSC proliferation and hepatic transforming growth factor (TGF)-ß1, α1(I)-procollagen, and tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA expression and hydroxyproline levels. Obeticholic acid inhibited hepatic TLR4 expression and increased hepatic matrix metalloproteinase-2 expression. Obeticholic acid decreased intestinal permeability by ameliorating CDAA diet-induced zonula occludens-1 disruption, whereas sitagliptin directly inhibited Ac-HSC proliferation. The in vitro suppressive effects of OCA + sitagliptin on TGF-ß1 and α1(I)-procollagen mRNA expression and p38 phosphorylation in Ac-HSCs were almost consistent. Sitagliptin directly inhibited the regulation of Ac-HSC. CONCLUSIONS: Treatment with OCA + sitagliptin synergistically affected hepatic fibrogenesis by counteracting endotoxemia induced by intestinal barrier dysfunction and suppressing Ac-HSC proliferation. Thus, OCA + sitagliptin could be a promising therapeutic strategy for NASH.

Combined treatment with dipeptidyl peptidase-4 inhibitor (sitagliptin) and angiotensin-II type 1 receptor blocker (losartan) suppresses progression in a non-diabetic rat model of steatohepatitis.

AIM: Dipeptidyl peptidase-4 (DPP4) inhibitors (DPP4-I) are oral glucose-lowering drugs for type 2 diabetes mellitus. Previously, we reported that DPP4-I (sitagliptin) exerted suppressive effects on experimental liver fibrosis in rats. Blockade of the renin-angiotensin system by angiotensin-II type 1 receptor blocker (losartan), commonly used in the management of hypertension, has been shown to significantly alleviate hepatic fibrogenesis and carcinogenesis. We aimed to elucidate the effects and possible mechanisms of a sitagliptin + losartan combination on the progression of non-diabetic non-alcoholic steatohepatitis (NASH) in a rat model. METHODS: To induce NASH, Fischer 344 rats were fed a choline-deficient L-amino acid-defined diet for 12 weeks. We elucidated the chemopreventive effects of sitagliptin + losartan, especially in conjunction with hepatic stellate cell (HSC) activation, angiogenesis, and oxidative stress, all known to play important roles in the progression of NASH. RESULTS: Sitagliptin + losartan suppressed choline-deficient L-amino acid-defined diet-induced hepatic fibrogenesis and carcinogenesis. The combination treatment exerted a greater inhibitory effect than monotherapy. These inhibitory effects occurred almost concurrently with the suppression of HSC activation, neovascularization, and oxidative stress. In vitro studies showed that sitagliptin + losartan inhibited angiotensin II-induced proliferation and expression of transforming growth factor-ß1 and α1 (I)-procollagen mRNA of activated HSC and in vitro angiogenesis, in parallel with the suppression observed in in vivo studies. CONCLUSIONS: The widely and safely used sitagliptin + losartan combination treatment in clinical practice could be an effective strategy against NASH.

Incidence of and risk factors for metachronous gastric cancer after endoscopic resection and successful Helicobacter pylori eradication: results of a large-scale, multicenter cohort study in Japan.

BACKGROUND: A previous multicenter prospective randomized study from Japan showed that Helicobacter pylori eradication reduced the development of metachronous gastric cancer (MGC) after endoscopic resection for early gastric cancer. MGC risk, however, is not eliminated; yet few studies have evaluated its long-term incidence and risk factors. In this study, we investigated the incidence of and risk factors for MGC in patients who underwent endoscopic resection for early gastric cancer with successful H. pylori eradication. METHODS: A total of 594 patients who underwent endoscopic resection for early gastric cancer and successful H. pylori eradication at three institutions (National Cancer Center Hospital, University of Tokyo Hospital, and Wakayama Medical University Hospital) were analyzed retrospectively. Annual endoscopic surveillance was performed after initial endoscopic resection. MGC was defined as a gastric cancer newly detected at least 1 year after successful H. pylori eradication. RESULTS: Ninety-four MGCs were detected in 79 patients during the 4.5-year median follow-up period. Kaplan-Meier analysis showed the cumulative incidence of MGC 5 years after successful H. pylori eradication was 15.0 %; the incidence of MGC calculated by use of the person-year method was 29.9 cases per 1000 person-years. Multivariate analysis using the Cox proportional hazards model revealed that male sex, severe gastric mucosal atrophy, and multiple gastric cancers before successful H. pylori eradication were independent risk factors for MGC. Eleven percent of MGCs (10 of 94) were detected more than 5 years after successful H. pylori eradication. CONCLUSION: Surveillance endoscopy for MGC in patients who have undergone endoscopic resection for early gastric cancer should be performed even after successful H. pylori eradication.

Demonstration of the usefulness of epigenetic cancer risk prediction by a multicentre prospective cohort study.

BACKGROUND: Epigenetic alterations accumulate in normal-appearing tissues of patients with cancer, producing an epigenetic field defect. Cross-sectional studies show that the degree of the defect may be associated with risk in some types of cancer, especially cancers associated with chronic inflammation. OBJECTIVE: To demonstrate, by a multicentre prospective cohort study, that the risk of metachronous gastric cancer after endoscopic resection (ER) can be predicted by assessment of the epigenetic field defect using methylation levels. DESIGN: Patients with early gastric cancer, aged 40-80 years, who planned to have, or had undergone, ER, were enrolled at least 6 months after Helicobacter pylori infection discontinued. Methylation levels of three preselected genes (miR-124a-3, EMX1 and NKX6-1) were measured by quantitative methylation-specific PCR. Patients were followed up annually by endoscopy, and the primary endpoint was defined as detection of a metachronous gastric cancer. Authentic metachronous gastric cancers were defined as cancers excluding those detected within 1 year after the enrolment. RESULTS: Among 826 patients enrolled, 782 patients had at least one follow-up, with a median follow-up of 2.97 years. Authentic metachronous gastric cancers developed in 66 patients: 29, 16 and 21 patients at 1-2, 2-3 and ≥3 years after the enrolment, respectively. The highest quartile of the miR-124a-3 methylation level had a significant univariate HR (95% CI) (2.17 (1.07 to 4.41); p=0.032) and a multivariate-adjusted HR (2.30 (1.03 to 5.10); p=0.042) of developing authentic metachronous gastric cancers. Similar trends were seen for EMX1 and NKX6-1. CONCLUSIONS: Assessment of the degree of an epigenetic field defect is a promising cancer risk marker that takes account of life history.

Association of gastric cancer risk factors with DNA methylation levels in gastric mucosa of healthy Japanese: a cross-sectional study.

Helicobacter pylori infection induces aberrant DNA methylation, and methylation levels of several specific marker genes in gastric mucosa are associated with gastric cancer risk. However, it is unclear whether gastric cancer risk factors are associated with methylation levels of marker genes in healthy individuals. We conducted a cross-sectional study of 281 Japanese cancer screenees aged 40-69 years with no history of H.pylori eradication therapy who responded to a validated food frequency questionnaire. DNA methylation levels of marker genes (miR-124a-3, EMX1 and NKX6-1) in gastric mucosa were quantified by real-time methylation-specific polymerase chain reaction. A multivariate beta regression model was used to investigate the association of pack-years of smoking and intakes of green/yellow vegetables, fruit and salt with methylation levels of marker genes. All analyses were stratified by H.pylori status. We found 2.5 to 34.1 times higher mean methylation levels among those with current H.pylori infection (n = 117) compared to those without (n = 164). After adjustment for potential confounders, we found increased levels of miR-124a-3 methylation according to pack-years of smoking and decreased levels of methylation according to green/yellow vegetable intake. We did not detect these associations among those without H.pylori infection. In conclusion, smoking habits and green/yellow vegetable intake were associated with DNA methylation levels in gastric mucosae of healthy individuals with current H.pylori infection. Our study suggests that these risk factors may modify the effect of H.pylori on methylation induction and maintenance in gastric mucosa.

Hereditary diffuse gastric cancer in a Japanese family with a large deletion involving CDH1.

Hereditary diffuse gastric cancer (HDGC), characterized by susceptibility to gastric signet ring cell carcinomas (SRCCs) and caused by CDH1 germline mutations, is rare in the Japanese. We present here a Japanese family with HDGC identified by comparative genomic hybridization (CGH) analysis. A 55-year-old woman was treated with completion gastrectomy for multiple SRCCs, and pathological examination revealed approximately 200 foci of SRCC with loss of E-cadherin expression. Her 30-year-old son had surveillance endoscopy and was found to have multiple SRCCs. He underwent total gastrectomy, and 32 foci of SRCC with loss of E-cadherin expression were histologically found. Although no point mutations were detected in CDH1 by sequencing, CGH revealed a 275-kb deletion involving exons 7-16 of CDH1 in both patients. While only a few HDGCs have been reported in East Asia, patients with multiple SRCC may need to be offered appropriate genetic counseling and testing in this area.

Laparoscopic Gastrectomy Using External Oblique Intercostal Block Versus Wound Infiltration: A Trial Protocol.

OBJECTIVE: A novel external oblique intercostal block (EOIB) might have analgesic effects on T6-10 and be indicated for laparoscopic gastrectomy. However, EOIB effects on postoperative pain are unknown. We aim to generate evidence to support such EOIB application. We will compare the efficacy of EOIB and wound infiltration (WI) in a single-center, single-blind, randomized controlled trial. METHODS: We will assess plasma concentrations of levobupivacaine after EOIB, its pharmacokinetics, and the pinprick test in patients randomly assigned to receive EOIB or WI before laparoscopic or robot-assisted gastric distal or total gastrectomy. The EOIB and WI will start after general anesthesia induction with 20 and 40 mL of 0.25% levobupivacaine per side, respectively, before skin closure. The outcomes will be numeric rating scale (NRS) scores at 12 h postoperatively (primary) and postoperative NRS scores at 2, 24, and 48 h; fentanyl application; QoR-15 scores on postoperative days 1, 2, and 7; and World Health Organization Disability Assessment Schedule 2.0 scores at 3 months (secondary). CONCLUSIONS: We hope that our study will provide evidence to support EOIB application in laparoscopic surgery. Plasma concentrations will help determine levobupivacaine pharmacokinetics, which if similar to conventional nerve blocks, will indicate EOIB's safety.

The clinical trial of alternative relugolix administration for uterine leiomyoma prior to surgically treatment: a study protocol for Non-Adverse Relugolix Administration (NARA) trial.

BACKGROUND: Uterine leiomyomas are common for reproductive-aged women and affect women's quality of life due to heavy menstrual bleeding or dysmenorrhea. Leiomyomas grow according to estradiol exposure and decrease after post-menopause. In case serious symptoms are caused by leiomyomas, pharmacotherapy or surgical treatment is proposed. Prior to surgical treatment, pharmacotherapies aimed at the reduction of leiomyoma and uterine volume or improvement of anemia are introduced to conduct minimum invasive surgery (i.e., to reduce blood loss or surgical duration). Recently, relugolix (40 mg orally once daily) as a gonadotropin-releasing hormone (GnRH) receptor antagonist has proved its sufficient efficacy in suppressing estradiol levels without the transient estradiol flare-up compared with GnRH agonist. However, long-term administration should not be permitted liable to for climacteric disorder or osteoporosis, and evidence is lacking on the actual efficacy and extent of adverse effects of the every-other-day dosing regimen. This trial aimed to prove non-inferiority in volume reduction effect on leiomyoma and safety (i.e., reduction of adverse effects) by every-other-day administration after 2 months of everyday administration compared to daily administration throughout the duration. METHODS: A minimization adaptive randomized control trial (RCT) will be conducted. Patients (over 20 years old) harboring leiomyoma who will be undergoing surgical treatment will be invited to participate. Patients who are enrolled in the intervention group will receive every-other-day administration for 16 weeks after 8 weeks of daily administration. Patients who are enrolled in the control group will receive daily throughout the 24 weeks. The primary outcome is the leiomyoma volume reduction, and the secondary endpoints are the reduction of uterine volume, the occurrence of the climacteric disorder, genital bleeding days, change rate of serum hormone or bone turnover markers, and bone mineral density after 24 weeks compared to before administration. DISCUSSION: This study aims to prove both the non-inferiority in leiomyoma volume reduction and superiority in adverse effects occurrence reduction, which will provide a novel method to escape adverse effects while maintaining the effect of leiomyoma reduction. TRIAL REGISTRATION: Japan Registry of Clinical Trials jRCTs051230078. Registered on 26 July 2023.

Clinical application of the CpG island methylator phenotype to prognostic diagnosis in neuroblastomas.

Clinical applications of aberrant DNA methylation to cancer diagnostics and therapeutics are accelerating. Especially, the CpG island methylator phenotype (CIMP), simultaneous methylation of multiple genes, provides information that cannot be obtained by other diagnostic methods and therapeutic opportunities. CIMP is known to be associated with poor or good prognosis depending upon cancer types. We identified that CIMP in neuroblastomas (NBLs) is strongly associated with poor prognosis in Japanese NBL cases (hazard ratio (HR)=22). Almost all NBLs with MYCN amplification displayed CIMP, and even among NBLs without MYCN amplification, NBLs with CIMP had worse prognosis (HR=12). The prognostic power was faithfully reproduced in German NBL cases by the same methods, and also in Italian and Swedish NBL cases with different analytical methods. Mechanistically, methylation silencing of different sets of tumor-suppressor genes is involved in poor prognosis of NBLs with CIMP, and the presence of CIMP is most sensitively detected by methylation of the PCDHB family. For therapeutic purposes, a combination of 5-aza-2'-deoxycytidine, a DNA-demethylating drug, with 13-cis-retinoic acid, a differentiating drug, has been shown to be effective for NBLs in vitro, and further development of a better combination(s) is awaited. Now, epigenetic diagnosis and therapeutics are becoming or have become an important choice for cancer patients.

Stronger prognostic power of the CpG island methylator phenotype than methylation of individual genes in neuroblastomas.

OBJECTIVE: The CpG island methylator phenotype is strongly associated with poor survival in neuroblastomas. Neuroblastomas with the CpG island methylator phenotype include almost all neuroblastomas with MYCN amplification, and, even among neuroblastomas without MYCN amplification, have worse prognosis. At the same time, methylation of individual tumor-suppressor genes is also reported to be associated with poor survival. The purpose of this study was to compare the prognostic power of the CpG island methylator phenotype with that of methylation of individual genes. METHODS: Methylation-specific polymerase chain reaction was performed for five individual genes (CASP8, EMP3, HOXA9, NR1I2 and CD44) in 140 Japanese and 152 German neuroblastomas. Kaplan-Meier analysis and log-rank tests were conducted to compare the survival between groups defined by methylation status. RESULTS: Among the five individual genes, only CASP8 methylation had a significant association with poor overall survival both in Japanese (hazard ratio = 3.1; 95% confidence interval = 1.5-6.4; P = 0.002) and German (hazard ratio = 4.8; 95% confidence interval = 2.1-11; P = 0.0002) neuroblastomas. HOXA9 and NR1I2 methylation were associated with poor survival only in German neuroblastomas. On the other hand, the CpG island methylator phenotype had a strong and consistent association in Japanese (hazard ratio = 22; 95% confidence interval = 5.3-93; P = 1.5 × 10(-5)) and German (hazard ratio = 9.5; 95% confidence interval = 3.2-28; P = 4.7 × 10(-5)) neuroblastomas. CONCLUSION: The CpG island methylator phenotype is likely to have stronger prognostic power than methylation of individual genes in neuroblastomas.

Identification of gastric cancer risk markers that are informative in individuals with past H. pylori infection.

BACKGROUND: Epigenomic damage induced by Helicobacter pylori infection is accumulated in gastric mucosae before the development of malignancy. In individuals without current H. pylori infection, DNA methylation levels of specific CpG islands (CGIs) are associated with gastric cancer risk. Because risk estimation in individuals with past infection is clinically important, we here aimed to identify the risk markers that reflect epigenomic damage induced by H. pylori infection, and that are informative in these individuals. METHODS: Gastric mucosae were obtained from 55 gastric cancer patients (GC-Pt) (21 with current infection and 34 with past infection) and 55 healthy volunteers (HV) (7 never-infected, 21 with current infection, and 27 with past infection). Hypermethylated CGIs were searched for by methylated DNA immunoprecipitation-CGI microarray, and methylation levels were analyzed by quantitative methylation-specific polymerase chain reaction (PCR). RESULTS: By microarray analysis of a pool of three samples from GC-Pt with past infection and another pool of samples from HV with past infection, 15 hypermethylated CGIs in the former pool were isolated. Seven of them had significantly higher methylation levels in GC-Pt with past infection (n = 10) than in HV with past infection (n = 10) (P < 0.001). In a validation cohort (21 GC-Pt with past infection and 14 HV with past infection), the seven new markers had large areas under the receiver-operating characteristic curves (0.78-0.84) and high odds ratios (12.7-36.0) compared with two currently available markers (0.60-0.65, 5.0-5.7). CONCLUSIONS: We identified seven novel gastric cancer risk markers that are highly informative in individuals with past infection.

Benefit of glucosyl Hesperidin in patients with primary biliary cholangitis: A multicenter, open-label, randomized control study.

INTRODUCTION: Globally, the number of patients with primary biliary cholangitis (PBC) is increasing. Growing evidence suggests that oxidative stress plays a significant role in the pathogenesis of chronic liver disease regardless of its etiology. Hesperidin, a natural antioxidative substance derived from citrus peel, has been shown to have an anti-inflammatory effect in a rat arthritis model and may be a potential substance to attenuate intrahepatic inflammation in patients with PBC. In this study, the potential of glucosyl hesperidin as a therapeutic agent for PBC will be investigated through antioxidative stress mechanisms. METHODS: Patients with PBC who are 20 years or older will be eligible to participate. Patients will be assigned to 1 of 2 groups and given either 500 or 1000 mg of glucosyl hesperidin per day. The primary endpoint is the ratio of changes in serum gamma-glutamyl transferase levels before and after 24 weeks of glucosyl hesperidin administration. The secondary endpoints are serum hepatobiliary enzyme levels (alkaline phosphatase, transaminase, and total bilirubin levels) and the protein expression levels of nuclear factor erythroid 2-related factor 2 and its target molecule 8, 16, and 24 weeks after administration compared to before administration. DISCUSSION: The prospective clinical interventional study was designed to assess the supportive effect of glucosyl hesperidin on hepatic function in patients with PBC receiving basic ursodeoxycholic acid treatment.

Feasibility and acceptability of novel functional electronic stimulated rehabilitation application for treatment in patients with cerebrovascular disorders: the FRAT study protocol.

BACKGROUND: The prognosis of patients with cerebrovascular disorders is poor owing to their high residual rate of hemiplegia. Delayed withdrawal from synkinesis is a major cause of prolonged hemiplegia; however, effective rehabilitation has not been established. This single-arm, open-label study aims to evaluate the influence of a low-frequency treatment device on canceling synkinesis in patients with incomplete paralysis and cerebrovascular disorders. METHODS: Eligible participants will include patients aged 20 years or older with incomplete paralysis, defined as upper limb Brunnstrom stage (BRS) of 2-4, who are within 1 month of onset of a cerebrovascular disorder. Qualified patients will be assigned to the novel rehabilitation treatment with IVES+ for 4 weeks. The primary endpoint of the study is the change from baseline in the upper-limb Fugl-Meyer Assessment (FMA) 2 weeks after the start of treatment. The secondary endpoints are changes in the amount of Functional Independence Measure, changes in the amount of upper-limb BRS, and changes in the amount of Barthel Index (BI) compared to the pre-intervention value at weeks 2 and 4; changes in the upper-limb FMA scores at 1, 3, and 4 weeks; changes in grip strength compared to the pre-intervention values at 1, 2, 3, and 4 weeks; and changes in upper-limb strength (manual muscle test) compared to the pre-intervention values at 1, 2, 3, and 4 weeks. DISCUSSION: This study will explore the usefulness of IVES+ for recovery from motor paralysis in patients with cerebrovascular disorders. TRIAL REGISTRATION: Japanese Clinical Registry, jRCTs052180226. Date of registration: February 1, 2022.

Effect of combined farnesoid X receptor agonist and angiotensin II type 1 receptor blocker on ongoing hepatic fibrosis.

OBJECTIVE: Nonalcoholic steatohepatitis (NASH) is difficult to diagnose in patients with no symptoms. We aimed to investigate the combined effect of farnesoid X receptor (FXR) agonist, obeticholic acid (OCA), and angiotensin II type 1 receptor blocker (ARB: losartan) on an ongoing hepatic fibrosis in a NASH rat model. METHODS: Fischer 344 rats were fed with choline-deficient L-amino-acid-defined (CDAA) diet for 16 weeks. After 8-week administration of CDAA diet, OCA, losartan, or a combination of these drugs was administered at a dose of 30 mg/kg/day for 8 weeks by oral gavage. The in vivo and in vitro effects of OCA + losartan and liver fibrosis progression, lipopolysaccharide (LPS), Toll-like receptor 4 (TLR4) regulatory cascade, and gut barrier function were evaluated. RESULTS: OCA + losartan alleviated hepatic fibrosis progression by suppressing α-SMA expression. It inhibited the proliferation of activated hepatic stellate cell (Ac-HSC) and mRNA expression of hepatic transforming growth factor-ß1 (TGF-ß1), TLR4, and tissue inhibitor of metalloproteinase-1 (TIMP-1) and decreased the hydroxyproline levels. OCA increased the hepatic matrix metalloproteinase-2 (MMP-2) mRNA expression. OCA decreased the mRNA expression of hepatic LPS-binding protein and intestinal permeability by ameliorating the disruption of CDAA diet-induced zonula occludens-1. Losartan directly inhibited the proliferation of Ac-HSC. The in vitro suppressive effects of OCA + losartan on the mRNA expressions of TGF-ß1 and α1(I)-procollagen, TLR4, and TIMP-1 in Ac-HSCs were almost in parallel. CONCLUSIONS: OCA + losartan suppressed the ongoing hepatic fibrosis by attenuating gut barrier dysfunction and suppressing Ac-HSC proliferation. Combined therapy may be a promising novel approach for NASH with fibrosis.

Clinical Significance of Serum Zinc Levels on the Development of Sarcopenia in Cirrhotic Patients.

Background/Aim: Sarcopenia increases the mortality in patients with cirrhosis. Approximately 60% of zinc is accumulated in skeletal muscle. We aimed to determine the role of subclinical zinc deficiency on sarcopenia development in patients with cirrhosis. Patients and Methods: We enrolled 151 patients with cirrhosis and divided them into the group with normal serum zinc levels (Group N: 80-130 µg/dl; n=38) and group with subclinical zinc deficiency (Group D: <80 µg/dl; n=113). The risk factors for sarcopenia were then investigated. Results: Group D had more sarcopenia cases than Group N (31.0% vs. 13.2%). In group D, HGS exhibited a weakly positive but significant correlation with serum zinc levels (R=0.287, p=0.00212), serum zinc levels negatively correlated with both ammonia and myostatin levels (R=-0.254, p=0.0078; R=-0.33, p<0.01), and low zinc levels were independently associated with sarcopenia development. Conclusion: Patients with cirrhosis showing subclinical zinc deficiency have a significantly higher risk of developing sarcopenia.

Aberrant DNA methylation in contrast with mutations.

Aberrant DNA methylation is known as an important cause of human cancers, along with mutations. Although aberrant methylation was initially speculated to be similar to mutations, it is now recognized that methylation is quite unlike mutations. Whereas the number of mutations in individual cancer cells is estimated to be approximately 80, that of aberrant methylation of promoter CpG islands reaches several hundred to 1000. Although mutations of a specific gene are very few in non-cancerous (thus polyclonal) tissues (usually at 1 x 10(-5)/cell), aberrant methylation of a specific gene can be present up to several 10% of cells. Mutagenic chemicals and radiation are well-known inducers of mutations, whereas chronic inflammation is deeply involved in methylation induction. Although mutations are induced in mostly random genes, methylation is induced in specific genes depending on tissues and inducers. Methylation is potentially reversible, unlike mutations. These characteristics of methylation are opening up new fields of application and research.

Novel oral plasminogen activator inhibitor­1 inhibitor TM5275 attenuates hepatic fibrosis under metabolic syndrome via suppression of activated hepatic stellate cells in rats.

An orally bioavailable small molecule inhibitor of plasminogen activator inhibitor­1 (PAI­1) is currently being clinically assessed as a novel antithrombotic agent. Although PAI­1 is known to serve a key role in the pathogenesis of metabolic syndrome (MetS) including nonalcoholic steatohepatitis (NASH), the pharmacological action of an oral PAI­1 inhibitor against the development of MetS­related liver fibrosis remains unclear. The current study was designed to explicate the effect of TM5275, an oral PAI­1 inhibitor, on MetS­related hepatic fibrogenesis. The in vivo antifibrotic effect of orally administered TM5275 was investigated in two different rat MetS models. Fischer 344 rats received a choline­deficient L­amino­acid­defined diet for 12 weeks to induce steatohepatitis with development of severe hepatic fibrosis. Otsuka Long­Evans Tokushima Fatty rats, used to model congenital diabetes, underwent intraperitoneal injection of porcine serum for 6 weeks to induce hepatic fibrosis under diabetic conditions. In each experimental model, TM5275 markedly ameliorated the development of hepatic fibrosis and suppressed the proliferation of activated hepatic stellate cells (HSCs). Additionally, the hepatic production of tumor growth factor (TGF)­ß1 and total collagen was suppressed. In vitro assays revealed that TGF­ß1 stimulated the upregulation of Serpine1 mRNA expression, which was inhibited by TM5275 treatment in cultured HSC­T6 cells, a rat HSC cell line. Furthermore, TM5275 substantially attenuated the TGF­ß1­stimulated proliferative and fibrogenic activity of HSCs by inhibiting AKT phosphorylation. Collectively, TM5275 demonstrated an antifibrotic effect on liver fibrosis in different rat MetS models, suppressing TGF­ß1­induced HSC proliferation and collagen synthesis. Thus, PAI­1 inhibitors may serve as effective future therapeutic agents against NASH­based hepatic fibrosis.